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Eur. J. Biochem.

236, 128-137 (1996)


0 FEBS 1996

Alanine metabolism in isolated human kidney tubules


Use of a mathematical model
Denis FOUQUE, Sylvie DUGELAY, Guy MARTIN, Jacqueline COMBET and Gabriel BAVEREL
Laboratoire de Physiopathologie Metabolique et RCnale, Institut National de la SantC et de la Recherche Mtdicale (CRI 95 02 Ol),
Facultt de MCdecine Alexis Carrel, Lyon, France

(Received 18 Septembed21 November 1995) - EJB 95 1533/1

To gain insight into the fate of alanine nitrogen and carbon taken up by the human kidney under
certain conditions, isolated human kidney cortex tubules were incubated in Krebs-Henseleit medium with
L-alanine as substrate. The tubules metabolized alanine at high rates and in a dose-dependent manner.
Most of the alanine nitrogen removed was recovered as ammonia and to a lesser extent as glutamate.
Glucose, lactate and glutamate were also found to be significant products of alanine carbon metabolism.
A simple mathematical model allowing one to calculate flux of alanine carbon through the various meta-
bolic steps involved is proposed and applied to data obtained in experiments in which 5 mM [U-'TI-,
[l-''C]-, [ 2 - ' T - and [3-'4C]alanine were used as substrates in parallel. About 4 0 8 of the alanine carbon
removed was recovered as CO, and oxidation of C1 of alanine accounted for most of the CO, released
from alanine. Calculations reveal that the ATP produced exceeded 3.2-fold the ATP consumed in relation
to alanine metabolism. It is concluded that, in human kidney, alanine may serve as an energy supplier
and as a precursor of glucose and ammonia.
Keywords: alanine; metabolism; human; kidney ; modeling.

On the basis of arteriovenous measurements, it is well docu- this condition [I31 and may also serve as an energy source for
mented that alanine, the main gluconeogenic amino acid in the renal transports. The mathematical model, whose basic principle
liver (see [I, 21 for reviews), is released in net amounts into the is the same as that utilized in a previous study 1141, and glossary
circulating blood by the human kidney under many physiologi- of symbols are detailed in the Appendix.
cal or pathological conditions. This has been demonstrated in
patients under normal acid base balance conditions [3, 41 and in
EXPERIMENTAL PROCEDURES
metabolic acidosis [3, 51 or in patients with diabetes mellitus
161, chronic renal failure [4] or alcoholic cirrhosis [7, 81. Alanine Preparation of isolated human kidney-cortex tuhules.
has also been shown to be synthesized by isolated human kidney Fresh normal kidney cortex was obtained from the uninkolved
tubules incubated with lactate or pyruvate as substrate 191 thanks pole of kidneys removed for neoplasm from 12-h-fasted adult
to the operation of alanine aminotransferase which is known to patients. Specimens of cortex were immediately dissected and
be present in human kidney tubules [lo, 1 I]. placed in ice-cold Krebs-Henseleit buffer gased with a mixture
It is also well established that, in obese patients undergoing of O,/CO, ( 1 9 : l ) until the beginning of the tubule isolation pe-
prolonged starvation, the kidney extracts circulating alanine in riod (usually within 10 min). Kidney tubules were prepared by
net amounts [12]. But, to our knowledge, the fate of the carbon collagenase treatment of renal cortex slices as described pre-
and nitrogen of alanine in human kidney has not yet been viously [9, 151.
studied. The present study was therefore undertaken to acquire Incubations. Incubations were performed for 30 and 60 min
information on alanine metabolism in isolated human kidney tu- at 37C in a shaking water bath in 25-ml stoppered conical
bules. For this, substrate utilization and product formation were flasks, each with a center well, with a mixture of 95% and
measured by enzymatic and radioactive methods, and a rela- 5 % CO, as gas phase. The tubules obtained were incubated in
tively simple mathematical model of alanine metabolism was 4 ml Krebs-Henseleit buffer pH 7.4 [16] containing as substrate
used to interpret the data obtained and evaluate the relative im- either 5 mM ~-[l-'"Clalanineor 5 mM ~-[2-"CC]aIanineor 5 mM
portance of the various pathways involved. ~-[3-'~C]alanine, o r 5 mM ~-[U-"C]alanine.
The results obtained show that human kidney tubules utilize In all experiments, each experimental condition was tested
alanine at high rates and convert its amino group mainly into in duplicate. Incubations were terminated by adding perchloric
lanine carbons contribute to the production acid (final concn 2%, by vol.) to each flask. In each experiment,
not only of glucose but also of CO,, lactate and glutamate. It is zero-time flasks with and without substrates, were prepared by
concluded that, in the kidney of subjects starved for prolonged adding perchloric acid before the tubules. Collection and mea-
periods, the alanine extracted may significantly contribute to the surement of the '"CO, released from ['"Clalanine were per-
enhanced production of glucose and ammonia observed under formed as described previously [17]. After removal of the dena-
turated protein by centrifugation (4000Xg for 10 min), the su-
Correspondence to G. Martin, FacultC de Mtdecine Alexis Carrel, pernatant was neutralized with 20% (mass/vol.) KOH for rnetab-
12 rue Guillaume Paradin, F-69372 Lyon cedex 08, France olite determination and isotope incorporation into C1 of gluta-
Fux: +33 78 77 87 39. mate.
Fouque et al. (Eur J. Biochem. 236) 129
Table 1. Metabolism of 1 mM and 5 mM L-alanine in isolated human kidney tubules. Kidney tubules were incubated as described in Experimen-
tal Procedures. Results for metabolite removal (-) or production are reported as means 5 SEM for four experiments. Each flask contained
2.5 i0.3 mg (for 1 mM alanine) and 4.7 i0.9 mg dry m i l s of tissue (for 5 mM alanine).

[Alanine] Incubation Metabolite removal (-) or production of


time _. -

alanine lactate pyruvate glutamate glucose ammonia


mM min pmol/g dry mass
-________
1 30 - 1 0 2 . 6 t 11.1 1 4 . 0 i 5.9 18.7 5 1.9 21.2 t 5.1 27.2 Z 5.5 126.4 ? 16.8
1 60 -220.0 2 20.7 30.8 i 3.8 31.4 f 2.5 23.7 -t 6.5 54.7 2 11.9 270.5 5 41.2
0 30 - 13.8 t 6.0 - 2.1 ? 2.1 1.9 i 1.4 - 9.0t 10.0 24.2 t 2.3 101.2 2 6.8
0 60 - 10.4 t 7.0 6.6 t 6.0 1.6 5 1.6 - 19.8 t 7.7 40.4 t 6.5 178.3 2 19.6
5 30 -301.0 f 33.8 35.4 5 5.9 14.7 5 2.8 65.8 t 7.6 29.1 t 3.8 246.9 2 18.7
5 60 -628.3 f 66.0 50.5 ? 9.2 10.7 -t 1.1 117.1 Z 10.1 63.3 t 11.7 512.0 2 40.7

Analytical methods and calculations. Aspartate, alanine, of intermediates of the tricarboxylic acid cycle or of glycogen
glutamate, glutamine, ammonia, pyruvate, lactate, glucose, gly- occurred. Considering that two alanine moleculec are needed to
cogen, intermediates of the tricarboxylic acid cycle as well as form one glucose or glutamate molecule, and that one alanine
the dry mass of the amount of tubules added to the flasks were molecule gives rise to one lactate or pyruvate molecule, it can
determined as previouly described [IS, 171. The specific activity be calculated from the data of Table 1 that more than half the
of L-[1-'4C]glutamate was measured by the action of glutamic alanine carbons utilized by the tubules was accounted for by the
decarboxylase as described by Squires and Brosnan [18]. carbon products accumulated.
Substrate utilization and product formation were calculated
as the difference between the total contents of the flask (tissue Metabolism of increasing concentrations of alanine in hu-
+ medium) at the start (zero-time flasks) and after the period of man kidney tubules. Table 2 reveals that alanine utilization
incubation. The metabolic rates, reported as means 2 SEM, are increased in a dose-dependent manner up to a concentration of
expressed as rate of substance removal or production/dry mass 3-4 mM. This was also true for the accumulation of ammonia,
of tubules. glutamate, pyruvate and lactate. By contrast, the synthesis of
The rates of release of ' T O , from radioactive alanine mole- glucose observed in the presence of alanine, which was not
cules were calculated by dividing the radioactivity found in CO, much higher than that found from endogenous substrates (see
by the specific radioactivity of the labelled alanine carbons de- Table 2), reached a maximum at much lower alanine concentra-
termined in the zero-time samples for each experiment. tions.
Reagents. L-Alanine and glutamic decarboxylase (grade V) In two other series of four experiments, it was found that
were from Sigma. Other enzymes and coenzymes were lactate was a better gluconeogenic precursor than alanine
purchased from Boehringer and all other chemicals were reagent (P<O.OOl): glucose synthesis was 136.7 -C 15.7 and
grade. ~ - [ l - ' ~ C ] A l a n i n(52
e Ci/mol) was from Amersham. Both 63.3-Cl1.7 pmol . h-' . g dry mass-' from 5 mM lactate and
[2-"CC]pyruvate (31.7 Ci/mol) and [3-'4C]pyruvate (30 Ci/mol) 5 mM alanine, respectively. The corresponding values were
were supplied by New England Nuclear. ~-[2-'~C]Alanine and L- 100.5 i 1 2 . 5 and 56.328.5 pmol . h-' . g dry mass-'
[3-'4C]alaninewere synthesized from the corresponding labelled ( P < 0.001) with 1 mM substrate concentration.
pyruvate in the presence of alanine dehydrogenase and stoichio-
metric amounts of NH4C1 and an excess of NADH; they were Metabolism of alanine by human kidney tubules in the pres-
then purified by column chromatography as described by other ence of other physiological substrates. Four experiments were
authors 119, 201. performed with human kidney tubules incubated for 60 min with
a near-physiological concentration (1 mM) of L-alanine alone or
in the presence of 1 mM L-glutamine, or 1 mM L-lactate, or
RESULTS 1 mM L-glutamine + 1 mM L-lactate; under these conditions,
Time course of the metabolism of 1 and 5 mM alanine in alanine utilization was 287.7 2 10.6, 260.9 -t 12.7 (not signifi-
human kidney tubules. Table 1 shows that alanine utilization, cant), 142.0 2 7.3 ( P < 0.01) and 99.6 2 13.9 ( P < 0.001) pmol .
which greatly increased with substrate concentration, was linear h-' . g dry mass-', respectively.
with time. Most of the alanine nitrogen removed was recovered
as ammonia whose release was also linear with time at both Metabolism of 5 mM [l-14C]-, [2-l4CI-, [3-l4C1 and [U-'4Cl-
substrate concentrations (1 mM and 5 mM). A significant alanine. Application of the model. Table 3 shows the results
amount of alanine nitrogen was found as glutamate which accu- obtained with specifically or uniformly labelled alanine; as ex-
mulated linearly with time at 5 mM but not at 1 mM alanine pected, the amount of alanine utilized and that of products
concentration. No glutamine or aspartate was formed from 1 or formed were not significantly different with the various species
5 mM alanine. of labelled alanine and, therefore only the averaged values are
The accumulation of glutamate plus the formation of ammo- reported in this table. Table 3 also shows that, as expected, the
nia were sufficient to fully explain the utilization of alanine ni- sum of the releases of l4CO2from [l-'"C]-, [2-I4C] and [3-14C]-
trogen at 5 mM but not l mM substrate concentration, which alanine was close to the release of ' T O , from [U-'"Clalanine,
suggests that the release of ammonia which occurs from endoge- which accounted for the alanine utilized and not explained by
nous substrates in the absence of added alanine (see Table 1) the non-volatile carbon products found as revealed by carbon
was virtually abolished upon addition of 5 mM alanine. balance calculations. The release of ''C0, from [U-'"C]alanine
Table 1 also shows that the alanine carbons were recovered indicates that about one third of the alanine carbon metabolized
as glutamate, glucose, lactate and pyruvate. No accumulation was oxidized and the release of 14C0, from [ 1-"C]alanine
130 Fouque et al. (Eur: J. Biochem. 236)

Table 2. Metabolism of various concentrations of L-alanine in human kidney tubules. Kidney tubules (6.8 t 0.9 mg dry masdflask) were
incubated for 60 min as described in Experimental Procedures. Results for metabolite removal (-) or production are reported as means t SEhl for
four experiments.

[Alanine] Metabolite removal (-) or production of

alanine glucose lactate pyruvate glutamate ammonia

mM pmol h-' g dry mass-'

0 9.90 2 21.3 40.6 t 5.0 4.0 i 3.0 2.8 t 0.8 - 27.7 t 0.7 140.6 t 6.9
0.2 - 21.2 t 3.6 47.5 t 5.9 7.5 t 4.4 4.6 t 0.8 - 23.2 t 2.8 167.1 t 10.8
0.4 - 78.4 t 6.8 52.9 t 4.9 10.0 5 3.2 5.7 t 1.1 - 18.0 t 5.4 219.5 i 9.9
0.6 -110.2 t 15.4 58.1 t 5.8 14.3 i 3.3 8.3 t 2.4 - 12.9 t 5.0 269.4 i 28.4
0.8 -153.6 t 15.4 61.6 t- 6.9 23.0 t- 4.0 10.2 t 3.2 - 4 . 0 t 5.0 289.5 i 19.1
1.o -213.6 t 15.9 63.1 ? 6.2 25.3 2 4.7 11.7 5 2.8 3.7 t 5.2 320.5 t 10.7
1.2 -244.9 t 12.0 65.1 t 5.3 29.9 ? 4.1 14.1 t 2.9 16.5 i 6.0 340.2 t 12.9
1.4 -293.6 t 9.7 66.2 t 5 9 33.5 t 4.6 14.4 t 3.3 23.3 t 7.3 376.9 t 26.5
1.6 -295.0 t 11.3 64.8 t 4.8 36.9 ? 4.9 17.1 i 3.4 34.1 i- 7.3 403.6 t 10.0
1.8 -312.9 t 10.0 65.9 2 5.3 40.1 t 5.1 17.0 t 3.7 45.5 t 7.5 414.3 t 12.2
2.0 -362.0 t 6.3 64.5 ? 5.0 43.4 t 6.2 18.0 t 3.9 51.4 t- 8.5 417.8 ? 21.0
3.0 -462.6 t 10.6 62.2 t 5.0 50.3 t 6.3 19.3 2 4.1 80.1 t 9.7 452.4 t- 26.9
4.0 -539.2 t 28.3 59.5 t 4.4 54.0 t 6.5 21.7 2 4.6 109.2 i 11.6 462.8 t 22.4
5.0 -538.8 t 81.4 55.9 t 3.8 57.8 5 5.2 23.4 t 3.8 127.5 i 9.7 486.0 t 27.6

Table 3. Metabolism of 5 mM L-[I-'~C]-,L-[~-'~C]-, L - [ ~ - ~ ~ and


C ] -~-[U'~C]alanine
in human kidney tubules. Kidney tubules (14.1 t 0.2 nig
dry mdsslflask) were incubated with 5 mM I&-alaninefor 60 min as described in Experimental Procedures. Results for metabolite removal i - ) or
production are reported as means t SEM for four experiments performed with tubules prepared from four different kidneys.

Labelled Metabolite removal (-) or production of


alanine
alanine pyruvate lactate glucose glutamate ammonia

pmol. h ' . g dry mass-'


Any -487.4 t 42.1 11.7 t 1.6 30.5 t 1.9 65.3 5 7.1 118.0 t 11.6 414.2 t 15.1

Labelled product release or accumulation

1 4 ~ 0 , I1 -'4C]glutamate

pmol . h-' . g dry mass-'

[U -"C] Alanine 580.3 t 50.8 19.5 2 1.9


[3-14C]Alanine 71.5 i 8.1 3.2 t 0.5
[2-'T]AIanine 110.3 t 11.4 5.6 t 0.5
[l -'"C]Alanine 355.7 t 24.8 8.5 i- 1.1

clearly shows that the C1 of alanine contributed to about two are shown in Tables 4 and 5, respectively. It appears from calcu-
thirds of the CO, released from alanine (see Table 3). The re- lation of c that only 50.8 2 1.0% of the pyruvate derived from
lease of I4CO2from [3-'4C]alanine, which was lower than that alanine was converted into oxaloacetate by the pyruvate cxbox-
from [2-"C]alanine (Table 3), gives the maximal amount of ala- ylase reaction (see the c value in Table 4 ) ; as indicated by the
nine carbon skeletons completely oxidized to CO,. As shown in d value in Table 4, 40.5 i- 1.1 % of the alanine removed was
Table 3, this complete oxidation represented only 14.7% of the converted into acetyl-CoA by the pyruvate dehydrogenase
alanine utilized by the tubules. It can also be seen in Table 3 reaction, and the remainder of the alanine used accumulated as
that a small proportion of the C1 of glutamate was found to pyruvate and lactate. The oxaloacetate synthesized by py-
be labelled not only when 12-'T]- and [3-l4CJalaninewere the ruvate carboxylase was mainly converted into citrate ( ( 1 =
substrates but also when [l-'4C]alanine was metabolized. This 0.68020.005) and the remainder ( b = 0.320-tO.005) mas di-
means that a small proportion of the [l-'4C]oxaloacetate synthe- rected to the synthesis of phosphoenolpyruvate and gluco5e (Ta-
sized from [I -'T]alanine underwent equilibration with fumarate ble 4)..0f the citrate synthesized, 57.3 -C 1.1 % (s) was recycled
to give subsequently [4-14C]oxaloacetatewhich leads to the for- to give oxaloacetate, while 42.7 -C 1.1 % (= I-s) was converted
mation of [l -'T]glutamate (see Fig. 2). into glutamate and, finally, 39.0 2 0.8 % of the oxaloacetate syn-
thesized by pyruvate carboxylase was recycled as indicated by
Parameters of alanine metabolism. The various proportions the value of g (see Table 4).
and fluxes through pathways of alanine metabolism, calculated It can also be seen in Table 4 that the inversion i resulting
from the results given in Table 3 as explained in the Appendix, from the equilibration of oxaloacetate with fumarate was small.
Fouque et al. ( E m J. Biochern. 236) 131
Table 4. Various proportions through pathways of alanine metabo- nearly 98% of the products was formed after four Krebs cycle
lism in human kidney tubules. Values, given as means t SE for four turns.
experiments, were calculated from those of Table 3. Lower-case letters Table 5 also presents the absolute values of the various
indicate the proportion of a given intermediate metabolized at a given fluxes calculated for all Krebs cycle turns. These values il-
step. The symbols of the various proportions, defined in the Appendix,
are shown in Figs 2-4. AcCoA, acetyl-CoA; Cit, citrate; Glu, gluta- lustrate again that a large amount of the alanine-derived pyruvate
mate; Lac, lactate; OAA, oxaloacetate; 2-OG, 2-oxoglutarate; P-pyr- wac metabolized by pyruvate carboxylase which provided a
uvate, phosphoenolpyruvate; Pyr, pyruvate. large fraction of the total oxaloacetate synthesized but, due to
oxaloacetate recycling, not all this accumulated. It was ascer-
Substrate Product Parameter tained that the specific activity of labelled alanine did not
decrease during incubation.
notation value

pmol h I .g I
DISCUSSION
OAA Cit a 0.680 t 0.005
OAA P-pyruvute b 0.320 2 0.005 The present study provides information on the characteristics
PYr OAA C 0.508 2 0.010 of alanine metabolism in isolated human kidney tubules. The
Pyr AcCoA d 0.405 ? 0.01 1 latter appear to be an experimental preparation of great value
PYr Lac 1 0.063 ? 0.005
2-OG 0.427 t 0.011 which has already been used in previous studies to characterize
Glu ( 1-1
2-OG OAA 7 0.573 5 0.011 lactate, pyruvate, glutamine and glutamate metabolism [9, 21 1.
PYr accumulated Pyr p 0.024 ? 0.001 Our observation that, at physiological concentrations, ala-
nine utilization by the tubules increased in a concentration-de-
Other parameters pendent manner strongly suggests that, in vivo, the human kid-
inversion of OAA i 0.117 2 0.008 ney may adapt its utilization of alanine to the circulating levels
recycling of OAA g = a.s 0.390 ? 0.008 of this amino acid. That the human kidney may utilize alanine
even in the presence of other important renal substrates such as
lactate and glutamine is strongly suggested by the data given in
Results. It is also conceivable that, under normal conditions, the
human kidney utilizes and releases alanine simultaneously as
Table 5. Fluxes through pathways of alanine metabolism in human does the dog kidney 1221. It is therefore of physiological and
kidney tubules. Values, given as means C SE for four experiments, were nutritional importance to identify the fate and the metabolic
calculated from those of Table 3; fluxes, represented by capital letters pathways of alanine nitrogen and carbons. However, the rele-
and defined in the Appendix, are shown in Fig. 1 and expressed as rates vance of our results, obtained in vitro with alanine as sole sub-
of accumulation or conversion of equivalent C, unitsldry mass of
tubules. strate, to the situation in vivo remains to be studied; one must
take into account the presence of other renal substrates that may
Flux Symbol Value considerably alter the metabolism of alanine.
Our data clearly establish that most of the alanine nitrogen
pmol . h-' . g-' was released as ammonia; this information, together with the
Accumulation of pyruvate P 11.7 2 1.6 presence of high activities of both alanine aminotransferase and
of lactate L 30.5 2 1.9 glutamate dehydrogenase in human kidney cortex [lo, 111, are
Conversion by Ala aminotransferase X 487.4 2 42.1 consistent with the view that alanine nitrogen was transferred to
by pyruvate carboxylase C 248.5 2 25.6 2-oxoglutarate by alanine aminotransferase to yield glutamate
by citrate synthase A 278 2 31.4 whose nitrogen was subsequently released as ammonia by gluta-
Accumulation of Glu A-G 118.0 2 11.6 mate dehydrogenase (see Fig. 1 in the Appendix). The observa-
Conversion by 20G dehydrogenase G 160.0 t 20.2 tion that glutamate accumulated when alanine was the substrate
by P-pyruvate carboxykinase B 130.5 t 14.2 is also consonant with the view that ammonia was released after
by pyruvate dehydrogenase D 196.7 C 14.7 prior synthesis of glutamate. These results suggest that, like in
the dog kidney [23], alanine contributes in human kidney to the
ammonia excreted in urine and therefore to the defense of sys-
temic acid/base balance. The absence of glutamine synthesis
from alanine fits with the previous demonstration that the human
Using the various equations presented in the Appendix, it kidney is devoid of glutamine synthetase activity [24].
has been possible to calculate fluxes through the different path- The present study establishes that, in human kidney tubules,
ways of alanine metabolism for infinite Krebs cycle turns (see unlike in guinea-pig kidney tubules [2S], alanine is a gluco-
Table 5). The results indicate that 28.1 -t- 4.2% of the acetyl-CoA neogenic precursor (Tables 1-3). This holds true not only at
needed for citrate synthesis was of endogenous origin because high concentrations, but also at low (physiological) concentra-
71.9?4.2% (= DIA) of this acetyl-CoA was provided by the tions (Table 2). This strongly suggests that, in human kidney
pyruvate dehydrogenase reaction. under prolonged starvation where glucose is released at rates
Table S shows that, during alanine metabolism, there was a similar to those in the liver [13], alanine, the main hepatic gluco-
large oxaloacetate synthesis (but without significant change in neogenic amino acid [ I , 21, contributes to glucose synthesis.
the total amount of oxaloacetate accumulated) resulting not only This view is supported by the calculations made by Owen et al.
from the operation of pyruvate carboxylase but also from re- who concluded that the amino acid carbons (measured as u-
cycling via the Krebs cycle. This raises the question of how amino nitrogen) extracted by the human kidney had to be con-
many Krebs cycle turns were needed to synthesize the products. verted into glucose to account for the glucose released into the
The model used allows one to calculate that, for n complete renal vein under this condition 1131.
Krebs cycle turns, the proportion of products synthesized The model used in the present paper provides a simple repre-
was equal to 1 -g". From this formula, it can be calculated that sentation of alanine metabolism, the basic principle of which is
132 Fouque et al. ( E m J . Biochern. 2361

the same as that used in a previous study in isolated rat hepato- cose (in C, units) from alanine was considered to consunie 4
cytes 1141. This model, in which the major steps of calculation and 6 mol ATP/mol, respectively. Finally, it was assumed that
are completly described, has been adapted to the parameters 1 mol ATP was needed to transport 3 mol sodium ions out of
measured in the present study. Accordingly, new equations have the renal tubular cells to prevent (thanks to the Na+/K+-ATPase
been introduced. The recent models of alanine metabolism used reaction) the intracellular accumulation of the sodium cotrans-
to interpret data obtained in liver preparations [26, 271 are much ported with the alanine taken up. Calculations from the data
more complicated because they are based on the use of specific given in Table 5 indicate that the ATP produced (4020 p o l
activities of intermediates so that total fluxes (flux of the added . h-' . g dry mass-') during alanine metabolism exceeded 3.2-
substrate plus flux of the endogenous substrates) through the fold the ATP needed (1242 pmol . h-' . g dry mass-') to synthe-
enzymatic steps studied can be calculated. size its non-volatile end products and to transport out of the cells
By contrast, in our model, which was designed to calculate the sodium taken up concomitantly with the alanine uptake. This
only fluxes related to the utilization of the added substrate indicates that a large fraction of the ATP produced by alanine
through its various metabolic pathways (i.e. flux of alanine car- metabolism was available presumably for ion transports unre-
bons plus flux of carbons of endogenous substrates that com- lated to alanine metabolism.
bined with alanine carbons), only the total radioactivity transfer- In summary, this study provides the first detailed information
red from the labelled alanine to intermediates or end products regarding not only the metabolic fate of alanine carbon and ni-
has to be taken into account. In this respect, our modeling ap- trogen but also the importance of the pathways involved in ala-
proach is less complete than the kinetic analysis mentioned nine metabolism in human kidney tubules. The results obtained
above but it has the advantage of being based on no assumption. thanks to the use of a mathematical model in combination with
An additional advantage of our model is that it can be applied enzymatic and radioactive measurements indicate that, in human
at any time point and irrespective of whether or not the experi- kidney, the alanine extracted from circulating blood may repre-
ments are performed under steady-state conditions. The simulta- sent a source of energy and serve as a precursor of the ammonia
neous use of [U-'TI-, [I-'"C]-, [2-I4C]-and [3-"CC]alanine was and glucose formed by this organ.
made possible thanks to the synthesis in our laboratory of [2-
' T - and [3-'"C]alanine, two labelled molecules not commer-
cially available. This approach permitted a good picture of the APPENDIX
metabolic fate and metabolic pathway of each alanine carbon
Mathematical model of alanine metabolism in human kidney
to be obtained in each experiment, which in turn provided the
tubules
numerous pieces of information necessary for applying the sim-
ple model developed in this study. Schematic representation of alanine metabolism. I n our
It should be stressed that, with this simple model, flux model the calculation of parameters (corresponding to ratios)
through various enzymes could be calculated. Of the oxaloace- and subsequently of enzymatic fluxes is based on the amount of
tate synthesized by pyruvate carboxylase most was converted products formed during alanine metabolism and on the fate of
into citrate, the remainder being converted into glucose. It the individual alanine carbons. A general representation cy'f ala-
should be mentioned that flux of alanine carbon through py- nine metabolism in human kidney cortex is given in Fig. 1. This
ruvate dehydrogenase was important but slightly lower than that figure shows the Krebs cycle and the different pathways in-
through citrate synthase, which means that a small fraction of volved in alanine metabolism. It also shows the main products
the acetyl-CoA needed for citrate synthesis originated from en- accumulated during alanine metabolism. The fates of the three
dogenous substrates, presumably fatty acids. carbons of alanine are represented in Figs 2 and 3. Fig. 4 ,,bows
It should also be underlined that, thanks to our model, it was the successive proportions allowing to calculate the amount of
possible to calculate that 39% ( = g) of the total oxaloacetate labelled oxaloacetates formed from alanine labelled on its car-
synthesized (but that did not accumulate) was derived from the bons 3 and 2.
Krebs cycle operation (see Table 4) and that four Krebs cycle
turns were sufficient to explain almost all (98%) the products Notations. (These are also explained in Table 4.) [Met]"" is
found. the total amount of the metabolite (Met) formed as a result of
An interesting finding is that alanine carbons, and especially alanine metabolism. [C,Met]C'+ is the amount of the metabolite
C1, were oxidized to CO, to a significant extent in our tubule (Met) formed, labelled on its carbon y (where 1 5 y 5 6) xising
preparation (see Table 3), suggesting that this amino acid may from alanine labelled on its carbon 2, where z is equal to 1 . 2 or
contribute to the production of energy by the human kidney. 3 because we used [I-'"Clalanine, [2-'"C]alanine and [Y"C]-
Thanks to our model of alanine metabolism, which allows one to alanine as labelled substrates.
calculate flux of carbons related to alanine metabolism through The abbreviations used are as follows: 2-OG = 2-oxoglutar-
various ATP-producing or ATP-consuming enzymes, it is pos- ate; AcCoA = acetyl coenzyme A ; Ala = alanine; Cit = ci-
sible to calculate the ATP balance related to alanine metabolism trate; Fum = fumarate; Glc = glucose; Glu = glutamate: Ict =
in our human tubule preparations. In these calculations, it was isocitrate; Lac = lactate; Ma1 = malate; OAA = oxaloxetate;
considered that flux through pyruvate carboxylase consumed P-pyruvate = phosphoenolpyruvate; Pyr = pyruvate; Suc =
I mol ATP/mol, flux through pyruvate, isocitrate, 2-oxoglutar- succinate.
ate, glutamate and malate dehydrogenases led to the production
of 3 mol ATPImol, and flux through succinylCoA synthetase and Calculations of the parameters of the model. The different
succinate dehydrogenase yielded 1 and 2 mol ATP/mol, respec- letters employed in our model to characterize proportions
tively. Flux through glutamate dehydrogenase was taken as the (lower-case letters) are defined in Table 4 and the symbols for
difference between flux through alanine aminotransferase and enzymatic fluxes (capital letters) are shown in Fig. 1 and defined
glutamate accumulation. Assuming that palmitate was the fatty in Table 5 . A proportion represents the relative amount of a
acid supplying the endogenous acetyl-CoA metabolized during given intermediate that is converted into the next one. At each
alanine metabolism, it was also considered that approximately bifurcation, the sum of all the proportions adds up to 1. An
4 mol ATP were produced during the formation of each mol enzymatic flux corresponds to the amount of the metabolite
acetyl-CoA from palmitate. The production of lactate and glu- formed by the enzyme of interest after a I-h incubation. When
Fouque et al. (EUCJ. Biochem. 236) 133
P-pyruvate 7Glc

cF2A1%pyrxLac
J. -u I
Cit " \ I L
.1
A]
/ 2-f9 *co2
/4
-'-ks)
C 12-OG

4
V
PYr
*Glc *c02 s Glc
OAA OAA
Fig. 2. Metabolic fate of the C l of alanine in isolated human kidney
tubules. The amount of C1-labelled alanine (C,Ala) utilized to form
I ~ * SUC pyruvate is represented by X pmol . h I . g~ I . The proportions p and l
Fig. 1. Alanine pathways in isolated human kidney tubules. Alanine of C1 -labelled pyruvate (C,Pyr) accumulates as pyruvate and lactate,
is converted into pyruvate by alanine aminotransferase (X pmol . h-I respectively, while the proportions c and d of C,Pyr are converted into
. g-') and subsequently accumulates as pyruvate ( P pmol . h-' . g- I) C1 -labelled oxaloacetate (C,OAA) and acetyl-CoA (AcCoA), respec-
and lactate ( L pinol . h-' . g - ' j or is converted either into oxaloacetate tively. This acetyl-CoA will condense with oxaloacetate formed during
(C pmol . h - ' . g-I) after fixation of CO, by pyruvate carboxylase or the different Krebs cycle turns. A proportion i of C,OAA is inverted
into acetyl-CoA ( D pmol . h - ' gg'j after its C1 is released as CO, into C,OAA. A proportion b of oxaloacetate and malate gives phospho-
by pyruvate dehydrogenase. After a partial equilibration with fumarate, eizolpyruvate which enters the gluconeogenic pathway at the end of
which glucose is accumulated. A proportion u of oxaloacetate forms
oxaloacetate gives phosphoeizolpyruvate ( B pmol . h-' g-') due to the
phosphoenolpyruvate carboxykinase reaction which releases C4 of oxa- citrate. 'The proportion (1 - s) of 2-oxoglutarate accumulates as gluta-
mate, while the proportion s is recycled to give oxaloacetate. A propor-
loacetate as CO,. Phosphoenolpyruvate enters the gluconeogenic path-
way to be converted into glucose. A fraction of the oxaloacetate formed tion represents the relative amount of a given intermediate that is con-
by pyruvate carboxylase ( A pmol h-I . g I) condenses with acetyl- verted into the next one. The relative amount of substrate (C,Ala) trans-
CoA, formed partially from endogenous fatty acids, to yield citrate. C6 formed into any labelled intermediate or end product is obtained by
of isocitrate is released as CO, by isocitrate dehydrogenase which gives multiplying the successive proportions found on the pathway from the
substrate to the intermediate or end product of interest. The amount of
2-oxoglutarate. 2-Oxoglutarate accumulates as glutamate [(A- C) pmol
intermediate formed or of end product accumulated is obtained by
. h-' . g '1 or is transformed by 2-oxoglutarate dehydrogenase into succi-
multiplying the corresponding relative amount by the amount (x)of la-
nyl-CoA (G pmol . h~ ' g-I) and subsequently into oxaloacetate (G
belled alanine utilized; with C, Ala as substrate, these labelled intermedi-
pmol h ' . g ' j . Ala = alanine; Pyr = pyruvate; Lac = lactate; Ac-
ates or end products can be formed only during the first Krebs cycle
CoA = acetyl coenzyme A; Cit = citrate; Ict = isocitrate; 2-OG = 2-
turn.
oxoglutarate; Glu = glutamate; Suc = succinate; Fum = fumarate;
Ma1 = malate; OAA = oxaloacetate; Asp = aspartate; P-pyruvate =
phosphoenolpyruvate; Glc = glucose.

the enzymatic reaction was reversible, the net flux was mea-
sured. P
The amount (in pmolh) of any given intermediate or end ~ 3 ~ y r
product formed from the substrate (alanine) can be calculated
by multiplying the amount of the substrate removed (X)by the (C30AA # C3Mal) czOAA d C2Mag C20AA C30AA
successive proportions of intermediates passing through the dif-
ferent pathways leading to the intermediate or end product of I \
interest.
Thus,
C3P-pyruvate
b
C2P-pyruvate
\
[MetlA'"= X . (the successive proportions from Ala to Met) ( I ) i
*Glc *Glc
Eqn (1) applies also for labelled metabolites.
Fig. 1 also shows that the alanine utilized (X)is converted
Cl0AA c40AA c20AA c30AA
into pyruvate and subsequently accumulated either as glutamate
(A-G), glucose (B), lactate (L) or pyruvate (P). Figs 2 and 3 Fig. 3. Metabolic fate of the C3 of alanine during the first cycle turn
show that the proportion of pyruvate which was accumulated in isolated human kidney tubules. Proportions are the same as those
and the proportions of pyruvate transformed into oxaloacetate, presented in Fig. 2, hut oxaloacetate recycled after one complete Krebs
acetyl-CoA, lactate are p , c, d and I, respectively (see also cycle turn remains labelled, indicating that the fate of C3 of alanine, in
Table 4); consequently, the sum of these proportions adds up contrast with the fate of C1 of alanine, requires more than one Krebs
cycle turn to be defined. The synthesis of oxaloacetate resulting from
to 1 :
the first Krebs cycle turn is considered to represent the beginning of the
p+c+d+l=l. (2) second turn.
134 Fouque et al. ( E m J. Biochern. 236)

By using Eqn ( 2 ) , it is possible to calculate the amount of By using Eqn (lo), it is possible to calculate the total amount
metabolites formed from alanine and then to compare them with of these metabolites formed and then to compare them with the
the enzymatic fluxes given in Fig. 1 . enzymatic fluxes given in Fig. 1 as follows:

[Pyr]"'" = X = Alanine aminotransferase flux. (3) [P-pyruvate]"'" = [Glc]"'" = Xcb/(l - g )


= C . b/(l - g) = B = phosphoenolpyruvate flux (1 1)
Pyruvate-derived [OAA]"'" = X c = C (4)
= glucose-6-phosphatase flux.
= Pyruvate carboxylase flux.
[tit]"';' = [ 2-OG]"'" = XCU/(1 - g ) = C . a/( 1 - g)
[AcCoA]"'" = X d = D = Pyruvate dehydrogenase flux. (5)
= A = citrate synthase flux (12)
[Lac]"" = XI = L = Lactate dehydrogenase flux. (6) = isocitrate dehydrogenase flux.

It should also be mentioned that accumulated [Glu]"'" = X C . ( a - g)/(1 - g ) C . ( a - g)/(l-g) = (A - G)


= glutamate dehydrogenase plus alanine aminotransferase flux.
= Xp
[PyrIA1" = P. (7)
(13)
From Eqns (2) and (4-7), it follows that Krebs-cycle-derived [OAA]""' = X c g / ( l - g )
= C . g/(l - g) = G (14)
C+D+L+P=X
= 2-oxoglutarate dehydrogenase flux.
and therefore
D =X-P- C-L. (8) As shown in Fig. 1 , the total amount of oxaloacetate formed,
[OAA]"'", results not only from the operation of pyruvatt: car-
Now, it shoud be stressed that the operation of the Krebs boxylase but also from the oxaloacetate synthesized b,y the
cycle, which results in a theoretically infinite number of turns, Krebs cycle:
allows the resynthesis of all the Krebs cycle intermediates.
Therefore, the oxaloacetate molecules formed by pyruvate car- [OAAIA'"= pyruvate-derived [OAA]"'"
boxylation yield new molecules of oxaloacetate in the Krebs +
Krebs-cycle-derived [OAA]"'" .
cycle. A complete turn of the Krebs cycle is considered to have
occurred as soon as the metabolite of interest of this cycle has Eqns (14) and (4) allow one to calculate the total oxaloace
been resynthesized once. The proportion of oxaloacetate resyn- tate formation :
thesized after one Krebs cycle turn is given by a . s (see Fig. 2 [OAA]"'" = X c / ( l - g) = C+G. (15
or 3) and corresponds to the recycling factor in the Krebs cycle.
For simplicity (see Fig. 4), Then, the pyruvate carboxylase flux

letg =a.s. (9) C = ([OAAIA'"- G ) . (16)


Then, the proportion ( R ) of oxaloacetate resynthesized dur- Thus, according to Eqn (8), the flux through pyruvate dehy-
ing an infinite number of Krebs cycle turns is given by: drogenase, D, can be calculated. In isolated human kidney tu-
bules, the oxaloacetate formed is directed only towards citrate
formation ( A ) and glucose formation ( B ); therefore,
A+B = [OAAIA'". (17)
Figs 2 and 3 show the proportions of metabolite conversion
occurring during the first Krebs cycle turn: a and b are propor- Replacing [OAA]"'"by (A + B ) in Eqn (16), it follows that:
tions of oxaloacetate converted to citrate and phosphoenolpy-
ruvate, respectively; s and (1 - s) are proportions of 2-oxoglu- C = (A +B - G ) o r C = ( A G) + B
- (1 8)
tarate converted to oxaloacetate and glutamate, respectively (see where (A - G) and B represent the glutamate and the glucose
also Table 4). These figures also show the formation of citrate accumulated (the sum of which corresponds to the oxaloacetate
and, subsequently, 2-oxoglutarate and oxaloacetate from py- synthesized by pyruvate carboxylase), respectively. At this point,
ruvate-derived acetyl-CoA. Such a schematic representation is g, the recycling factor in Krebs cycle, should be calculated to
useful to follow the fate of labelled carbons of alanine. It should obtain the value of the enzymatic fluxes related to alaniiie me-
be mentioned here that the formation of a citrate molecule re- tabolism. The calculation of g requires one first to determine the
quires the condensation of an oxaloacetate molecule and of the amount of labelled CO, formed from alanine labelled on its car-
acetyl moiety of an acetyl-CoA molecule. The latter molecule bon 3 and 2, [4:C02]C;A'.'and (*CO2]'2*la, respectively Fig. 4
has two possible origins since it can be formed either from the (top) allows one to calculate [C20AA]'7"''', the total amount of
added alanine or from endogenous substrates. This is why the C2-labelled oxaloacetate formed from C3-labelled alanine as
formation of AcCoA is not included in the calculation of the follows. Let [C,OAA]?:& represent the amount of <',OAA
amounts of Krebs cycle intermediates formed to avoid to take formed from C,Ala at the beginning of the first Krebs cycle turn.
into account twice the same molecules.
Then, by using Eqn (I), one can calculate the theoretical = Xci
[C,OAA]~~fi;:I~, + X d ~ / 2= (1/2) . X +
. ( 2 i ~ d\).
amount of Krebs cycle intermediates and related metabolites
formed during the first Krebs cycle turn. These amounts of phos- Since a proportion (g/2) of C,OAA is resynthesized at each
phoenolpyruvate, glucose, citrate, 2-oxoglutarate, glutamate and Krebs cycle turn, it follows that the total proportion of C,OAA
oxaloacetate formed are Xcb, Xcb, Xcu, X c a , X c a (1 - s ) and resynthesized is 1/[1 - (g/2)] or 242 - g). Then,
Xcus, respectively. Since, as already indicated, a . s is equal to
g (see Eqn 9), X c a . (1 - s ) and X c a s are equal to X c . ( a - g)
[C,0AA]r7A'.'= [C,OAAI~.f& /[I - (g/2)] (19)
and Xcg, respectively. = x ' (2ic + ds)/(2 -g).
Fouque et al. ( E m J. Riochem. 236) 135

n=O
Fig. 4. Label incorporation into oxaloacetate from C3- and C2-labelled alanine. Top: the various proportions of labelled oxaloacetate obtained
in the presence of C3-labelled alanine over three Krebs cycle turns. The fates of oxaloacetate labelled from acetyl-CoA or oxaloacetate are assumed
to be the same. The repetitiveness of this diagram allows one to deduce the amount OP label incorporated into oxaloacetate for the following Krebs
cycle turns. The proportion g/2 represents a s/2 (see Fig. 2). Bottom: the various proportions of labelled oxaloacetate obtained in the presence of
C2-labelled alanine at the beginning of the first Krebs cycle turn.

Fig. 4 also shows that: Fig. 4 also shows that [C,OAA]C~Al"


= (g/2) . [C20AA]C2A'n
= (g/2) . [C20AA]'+'"
[C,OAA]C~A'" + XC . (1 - i) + X d ~ / 2 + Xc . i and
[C,OAA]'2*'" = [C,0AA]'2A'" (24)
and
[C,OAA]'2A'a = [C,OAA]'xA'" = (g/2) . [C,OAA]C'Al".
= (g/2) . [CIOAA]C2AIa X . d ~ / 2 +
(20)
where (g/2) . [C,0AA]C2A1" represents the amount of CIOAA or
Fig. 2 shows that oxaloacetate-derived labelled CO, is equal
C,OAA derived from C,Ala that has passed through the stage
to C1-labelled oxaloacetate multiplied by a , plus C4-labelled ox-
of C,OAA (see the top of Fig. 4), and X . ds/2 represents the
aloacetate multiplied by ( b + g ) (see also Fig. 2 legend), where
amount of C,OAA or C,OAA derived from C,Ala that has
g = a . s (see Eqn 9). Therefore,
passed through the stage of C,AcCoA (see the bottom of Fig. 4).
. a + [C,OAA]clA'a . (b + g )
[*CO2]C+IU = [CIOAA]C~A'" As indicated previously, one can calculate the formation of la-
= [C,0AA]c3A'" . ( a b g) . + + belled CO, from Fig. 2:

And, since oxaloacetate yields only either citrate or phospho- [*CO,]CZA]"= [C,0AA]C2A'". (1 + g). (25)
enolpyruvate, the sum of the proportions a and h adds up to I . Combining Eqns (23-25), we obtain:
Therefore :
= X . (1
[*C02]C2AId + g) . (C . (g/2) . [i (26)
(1 + g ) .
[*C02]C2AIa= [CIOAA]C,A1". (21 )
+ (1 - i) . g/(2 - g)] + ds/2} .
Combining Eqns (19-21), we obtain:
Let
[*CO,]CWa X (g/2) (1 + g ) (22)
1 '
w = [*CO,]'?^'" + [*C0,]"2"', (27)
'
(C ' [(l - i) + i.g/(2 - g)] + dd(2 - g ) } .
then Eqns (27, 22 and 26) yield:
Fig. 4 (bottom) allows one to calculate [C,OAA]C?A1", the to-
tal amount of C2-labelled oxaloacetate formed from C2-labelled w=x ' (cg + ds) ' (1 + g)/(2 - g ) . (28)
alanine as follows. Let [C,OAA]F$$, be the amount of C,OAA Let
formed from C,Ala at the beginning of the first Krebs cycle turn. t = W/[X . (cg + ds)] (29)
[C,OAA]~$&, = Xc . (1 - i ) . then from Eqns (28) and (29), one can calculate that t = (1 + g)/
Since, as previously mentioned, a proportion (g/2) of C,OAA is (2 - g ) and deduce that
resynthesized at each Krebs cycle turn, it follows that the total g = (2t - 1)/(1 + t ) . (30)
proportion of C,OAA resynthesized is 1/11 - (g/2)] or 242 - g).
Then : Rewritting Eqn (29) yields a new equation allowing one to
[C20AA]C2A'a = [C2OAAI%%/ (23) calculate W as a function of t and g :
I1 - (g/2)] = 2 . X c . (1 - i)/(2 - g ) . w = [X (cg + ds)]
' ' t. (31)
136 Fouque et al. (ELKJ. Biochem. 236)

Since t is not a parameter independent from g (see Eqn 29), A = (A-G) + G = flux through citrate synthase Eqn (32)
g is calculated by iteration as follows: t is given an arbitrary
value and the final value of t and, subsequently, g is obtained
a = A/(C + G) = proportion of oxaloacetate giving citrate
Eqns (12) and (15)
when the calculated W fits with the measured W As soon as g
is known, Eqn (14) allows one to calculate the 2-oxoglutarate b = B/(C + G) = 1 - a
dehydrogenase flux, G, as a function of pyruvate carboxylase = proportion of oxaloacetate giving glucose
flux, C , which is already known (see Eqn 18). As glutamate Eqns (11) and (15)
accumulation (A - G) is measured in our experiments, the deter-
s = gla
mination of G allows one to calculate flux through citrate syn-
= proportion of 2-oxoglutarate converted into oxaloacetate
thase (A) as follows:
E w (9)
A = (A - G)+ G . (32) Calculated W = [X . (cg + ds)] . t . Eqn (3 1)
Fig. 2 shows that, in the presence of CI-labelled alanine, C1- Until the calculated and experimental W are equal, t is given a
labelled glutamate is formed exclusively during the first Krebs new arbitrary value allowing the calculation of a new value for
cycle turn, and allows one to calculate the corresponding g and the subsequent parameters necessary to calculate W.
amount:
[C,G~U]CIA'~'= X . c . i . a . (I - s ) . (33) X - ( A- G) = flux through glutamate dehydrogenase during all
the Krebs cycle turns (see Fig. 1 )
Fig. 2 also shows that the total amount of glutamate formed
during the first Krebs cycle turn is: i = proportion of oxaloacetate whose carbons have been in-
verted as a result of equilibration with fumarate. Eqri (36)
= X . c . u . (1
[G~u]~$:;',,,, - S) . (34) It should be stressed here that the calculation of g and of
Another formulation of the latter value is obtained by the subsequent parameters, as indicated in the above calculation
multiplying the total amount of glutamate formed by (1 - g) (see strategy, were done from an arbitrary value of t. The lattcr pa-
Fig. 1 and Eqn 10): rameter is complex and depends mainly on a measured par-ame-
ter W which is equal to the sum of [*C02]clAlaplus [*C071'~AL".
[G~U]?,':~~~~
= (A-G) . (1 - g) . (35) Thix parameter t was introduced only to obtain a more simple
From Eqns (33-35), one can calculate that: presentation of the calculations.

i = . c
[C,GILI]'~~'"/X .u . (1 - s). (36) The work was supported by the Institut Notional de la Sante et de
lo Recherclze Midicule (CRI 950201) and the University of Lyon 1.

Calculation strategy of the various parameters of alanine


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